Improving thermal hysteresis activity of antifreeze protein from recombinantPichia pastorisby removal of N-glycosylation

Abstract

To survive in a subzero environment, polar organisms produce ice-binding proteins (IBPs). These IBPs prevent the formation of large intracellular ice crystals, which may be fatal to the organism. Recently, a recombinant FfIBP (an IBP from Flavobacterium frigoris PS1) was cloned and produced in Pichia pastoris using fed-batch fermentation with methanol feeding. In this study, we demonstrate that FfIBP produced by P. pastoris has a glycosylation site, which diminishes the thermal hysteresis activity of FfIBP. The FfIBP expressed by P. pastoris exhibited a doublet on SDS-PAGE. The results of a glycosidase reaction suggested that FfIBP possesses complex N-linked oligosaccharides. These results indicate that the residues of the glycosylated site could disturb the binding of FfIBP to ice molecules. The findings of this study could be utilized to produce highly active antifreeze proteins on a large scale.

Precision cellular agriculture: The future role of recombinantly expressed protein as food

“…Conversely, the expression of prokaryotic AFPs in a eukaryotic host may result in unintended glycosylation. The eukaryotic expression system may recognize glycosylation sites that would natively be ignored during expression in a prokaryotic host (Kim et al., 2017). In the case of the Flavobacterium frigoris AFP, Ko.…”

“…phaffii added glycans at a recognized glycosylation site (Asn‐Met‐Ser), which was on the ice‐binding face of the protein, and hindered function. Mutations of the Asn in question (Asn203Ala, Asn203Gln) yielded proteins with larger thermal hysteresis values, albeit at the cost of secretion as a result of the loss of a glycosylation site (Kim et al., 2017). AFPs also display pH‐dependent function, although the mechanism for this is unclear (Xiao et al., 2010).…”